CN1839680A - Method for transferring Sichuan pickle using TuMV Hc-Pro resistant gene - Google Patents
Method for transferring Sichuan pickle using TuMV Hc-Pro resistant gene Download PDFInfo
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- CN1839680A CN1839680A CN 200510062200 CN200510062200A CN1839680A CN 1839680 A CN1839680 A CN 1839680A CN 200510062200 CN200510062200 CN 200510062200 CN 200510062200 A CN200510062200 A CN 200510062200A CN 1839680 A CN1839680 A CN 1839680A
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Abstract
This invention discloses a method for obtaining turnip mosaic antiviral preserved szechuan pickle by means of Hc-Pro gene inversion, the method takes twice subcultured cotyledon inducted wound-healing tissue as inversion receptor, and co-culture the tissue with meloidogynosis peasant bacillus culture liquid with Hc-Pro gene, then continuously sieve the inversed wound-healing tissue on antibiotics added medium, last do differentiation and replantation. Compared with current technics, the beneficial effects of this invention are:Hc-Pro gene inversion is process on cell, and this can improve inversion efficiency and inheritance stability of the gene transferred plant, and can obtain Hc-Pro gene taking plant instead of chimera gene transferred plant.
Description
Technical field
The present invention relates to a kind of bioengineering field, particularly a kind of method of transferring Sichuan pickle using TuMV Hc-Pro resistant gene.
Background technology
Hot pickled mustard tube is one of important vegetable of China, and it sells well all over the country, and finds a good sale in all over the world.Virus disease is the main disease of hot pickled mustard tube, takes place commonplace.Virus disease caused by Brassica 2 et 4 and cucumber mosaic virus, and wherein Brassica 2 et 4 (TuMV) is one of distinct issues the most.In the historical popular time, the average diseased plant rate of large tracts of land is more than 20%, and grave illness area and field piece are up to more than 50%, even full Tian Caitou does not have receipts.Take place in the main hot pickled mustard tube producing region of China in recent years year after year, the trend that increases the weight of is year by year more arranged.Therefore, various places require the cry of solution stemmustard disease viral disease quite strong, and it is particularly urgent and important that the cultivation of hot pickled mustard tube viral diseases material seems.
Plant thremmatology studies show that, can obtain the novel anti-virus kind by traditional sexual hybridization method, is anti-source material but at first require the parent who is used to hybridize.And studies show that in a large number: hot pickled mustard tube comprises all resistant materials of unmatchful TuMV of all kinds of leaf mustard, strain.We have carried out, and long-term leaf mustard germ plasm resource is collected and the screening of disease-resistant variety, fails to find the good resistance source material so far.Therefore, from natural resources, seek anti-source material, cultivate disease-resistant variety and seem difficult.
The appearance of plant gene engineering technology has brought new hope for the control of virus disease.With the nucleic acid composition of plant virus or with virus is the dna fragmentation importing host plant of effect target, has successfully obtained virus infection is had the plant of resistance.For example with virus capsid protein (Coat Protein, CP) gene changes tobacco over to, has obtained the antiviral gene tobacco of delayed onset, this research has caused that all over the world scientist pays close attention to.Therefore the research that transforms coat protein (CP) gene also obtained progress greatly.But with regard to its antiviral mechanism, most of viewpoint is to come from the disease resistance resistance that pathogen obtains, and promptly in the circulation of virus infection plant, the viral gene product was expressed with unfavorable quantity and mode in unfavorable time, thereby the infection ability of viral interference.But the CP gene is DeGrain on some crops, even does not have.Molecules in recent years studies show that, plant is not because the result of overexpression foreign gene to the resistance of virus, but mediated by the RNA molecule in the post-transcriptional silencing production process, therefore also is referred to as the virus resistance of RNA mediation.Hc-Pro (helper component-proteinase) is one and infects the multifunctional protein that works in the circulation at marmor upsilon, a class RNA viruses that belongs to Potyvirus, therefore, the Hc-Pro gene is more direct than the effect of CP gene, and effect is more obvious.By on hot pickled mustard tube, transforming the Hc-Pro gene, can obtain the hot pickled mustard tube material of resisting turnip mosaic virus, this is significant to hot pickled mustard tube production and germ plasm resource innovation.
Summary of the invention
The objective of the invention is to overcome weak point of the prior art, provide a kind of genetic transformation of Hc-Pro efficiently hot pickled mustard tube to obtain the method for resisting turnip mosaic virus.
In order to address the above problem, the present invention is achieved by the following technical solutions: a kind of method by Hc-Pro genetic transformation acquisition resisting turnip mosaic virus hot pickled mustard tube may further comprise the steps successively:
(1) callus that will induce through the cotyledon of 2 successive transfer culture is as transformation receptor;
(2) the Agrobacterium tumefaciems culture fluid and the callus that will have the Hc-Pro gene cultivated altogether;
(3) callus after will transforming constantly screens being added with on the antibiotic medium, breaks up then and plant regeneration.
2 successive transfer culture in the step (1) may further comprise the steps successively:
(1) preserved szechuan pickle seeds is stirred sterilization 1min with running water flushing back with 75% medicinal alcohol, in the 1%NaClO aqueous solution, stir 15min behind the distilled water flushing 3 times, aseptic water washing 4-5 time, on the superclean bench seed is being sowed on the MS+0.1mg/L BA sowing medium, is cultivating in 23-25 ℃ of down dark place.
(2) the 16h/d continuous illumination under same temperature of germination back is cultivated.
(3) the aseptic seedling cotyledon of 8 days seedling ages being cut into 2mm left and right sides small pieces puts into callus inducing medium 23-25 ℃ of dark place and cultivates.
Step (2) is carried out common cultivation 2 days under 24 ℃.
Compared with prior art, the invention has the beneficial effects as follows: the conversion of Hc-Pro gene is carried out on cellular level, has improved the genetic stability of transformation efficiency and transfer-gen plant, has avoided the generation of chimeric transfer-gen plant; What obtain is the Hc-Pro gene plant that has.
Description of drawings
Fig. 1 contains polyphone Hc-Pro gene and antiweed Bar gene for plasmid of the present invention;
Fig. 2 is that the molecules of transformed plant of the present invention is identified;
Among Fig. 2: 1-Marker, 2-contrasts (not having any template), the 3-plasmid DNA, 4-contrasts (non-transforming DNA), 5-8-transformant DNA;
Fig. 3 is that the molecules of transformed plant of the present invention is identified;
Among Fig. 3: 1-Marker, 2-contrasts (not having any template), the 3-plasmid DNA, 4-contrasts (non-transforming DNA), 5-8-transformant DNA;
Fig. 4 is that field of the present invention inoculation is identified and ELASA analyzes;
Among Fig. 4: the CK1-negative control; The CK2-positive control; CK3, the contrast of CK4-orthodox material; CK5, CK6-transform the no genes of interest materials control in back; 1-5-contains the genes of interest material.
Embodiment
Method key step of the present invention is: at first by inducing cotyledon to obtain the transformation receptor callus, infect callus with the Agrobacterium tumefaciems that has the Hc-pro gene, the callus that Agrobacterium was infected is put into common culture medium and is carried out common cultivation then.Afterwards, callus is through screening, break up and the final regeneration plant that obtains of taking root.
Embodiment 1:
Hot pickled mustard tube male sterile line and maintenance with selfing for many years in the present embodiment are material, get 7-8 days the inducing of aseptic seed young plant Ye Jinhang callus.
Employed Agrobacterium tumefaciems in the present embodiment (Agrobacterium tumefaciens) bacterial strain is EHA105 (Plh-IR) (available from a biological institute of Zhejiang University).Plasmid contains polyphone Hc-Pro gene and antiweed Bar gene (as Fig. 1).
Inducing of callus: washing afterwards with running water through selfing preserved szechuan pickle seeds for many years (vegetables institute of Zhejiang University) of full seed stirred sterilization 1min with 75% medicinal alcohol, in the 1%NaClO aqueous solution, stir 15min behind the distilled water flushing 3 times, aseptic water washing 4-5 time is being sowed at seed on the MS+0.1mg/L BA sowing medium on the superclean bench.The germination back 16h/d continuous illumination under same temperature of 23-25 ℃ of dark down place is cultivated.The aseptic seedling cotyledon of 8 days seedling ages is cut into 2mm left and right sides small pieces to be put into 23-25 ℃ of dark place of callus inducing medium (I) and cultivates, every the 16d subculture once, as transformation receptor, the total time that callus has been cultivated during conversion is 48 days with the callus of subculture 2 times.
The preparation of engineering bacteria liquid and infecting: the single colony inoculation of picking Agrobacterium EHA105 (Plh-IR) contains in the YEB liquid culture medium of the grand enzyme element of 100mg/L in 15mL, and 28 ℃ of following shaken cultivation are spent the night.Be diluted to OD with infecting the Agrobacterium culture of medium (III) in second day with incubated overnight
600Be about 0.1, in order to infect the hot pickled mustard tube callus after the common cultivation.The callus that subculture is 2 times soaks 10min in bacterium liquid, during constantly under 28 ℃ with the 100rpm velocity fluctuation, soak time is 10min.
The concrete operating procedure of Hc-Pro genetic transformation Agrobacterium EHA105 (Plh-IR) is according to (Methodsin Enzymology), the method that 1987.153:292-305 provided.
Agrobacterium and explant are cultivated altogether: the callus that Agrobacterium was infected is put into common culture medium (IV) and carry out common cultivation 2 days under 24 ℃.
Screen, break up and take root: after the cultivation, callus is dispersed in the middle screening twice of the screening culture medium (V) that is added with selection pressure (BASTA and cephalosporin) altogether, forwards middle differentiation of differential medium (II) of the cephalosporin that is added with 400mg/L then to.The bud that differentiates grows to 3-4 sheet true leaf in being added with 300mg/L cephalosporin 1/2MS medium, and then (VI) takes root in the root media that is added with the 300mg/L cephalosporin.Above cultivation temperature is about 25 ℃, and inducing and screening all and carry out under dark condition of callus begins to enter illumination cultivation from differentiation of calli, and intensity of illumination is 50 μ mols
-1M
-2, light application time is 16h/d.
The various medium of table 1 are formed
| Type of culture medium | Composition |
| Inducing culture I | MS minimal medium+0.25mg/L NAA+0.25mg/ |
| Differential medium II | MS minimal medium+0.2mg/L NAA+2mg/L BA |
| Infect medium ii I | The 1/2MS minimal medium (liquid, no agar, pH5.2) |
| Be total to culture medium IV | MS minimal medium+0.25mg/L NAA+0.25mg/ |
| Screening culture medium V | I type medium+0.15%BASTA+400mg/L cephalosporin |
| Root media VI | 1/2MS minimal medium+0.1mg/L NAA |
The molecules of transformed plant is identified:
Molecular biology identification: the plant leaf extraction DNA that chooses in the present embodiment by callus regeneration carries out PCR and Sounthern blot detection.
After testing, in transformed plant, increased segment (Fig. 2, Fig. 3) with plasmid DNA gene Hc-Pro of the same size and selection markers gene Bar.And same segment is not at non-transformed plant and do not contain in the amplification system of any template and obtain.Hence one can see that, and the T-DNA of plasmid has entered the genome of hot pickled mustard tube acceptor.
Field inoculation is identified and ELASA analyzes: get the tobacco young leaves of the existing obvious reveal any symptoms of the Brassica 2 et 4 inoculation that purifying crosses, use phosphate buffer milling and extracting virus.The method of extract with frictional inoculation is inoculated on hot pickled mustard tube (contrast and the transformant) young leaves.Be positioned over the greenhouse that isolates aphid.Get the contrast of inoculation Brassica 2 et 4 10d and young leaves 1 gram of converting material, carry out ELASA and analyze.The result shows that the plant that contains genes of interest is obviously on the low side than adjoining tree Brassica 2 et 4 content, though do not reach complete feminine gender, virus disease is had certain resistance, and its virus disease symptom performance contrast has than big-difference (Fig. 4).
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ ID NO：1
1 gtcacaagat tgtgcacttt agtgcagcag gagccaactt ctggaaaggc tttgacagat
61 gttttctcgc ataccgtagt gacaatcgcg agcatacatg ctattcaggg ttagatgtta
121 ctgagtgcgg cgaagtggca gcactgatgt gtttggctat gttcccatgc ggaaagataa
181 cctgccctga ctgtgtaaca gatagtgagc tatcccaagg acaagcaagc ggaccatcta
241 tgaagcacag gttaacgcag ctgcgcgatg tcatcaagtc aagctaccca cgcttcaagc
301 atgcagtgca gatactggac aggtatgagc aatcactgag cagtgcaaac gagaactacc
361 aagatttcgc agaaatccag agtataagcg atggagttga gaaagctgca ttcccacacg
421 tcaacaagct aaacgcaata ttgatcaaag gggccacagc gacaggggag gaattctcgc
481 aggccacgaa gcatttactc gagatagcac gatacctgaa gaacagaact gagaacattg
541 agaagggttc actgaagtcc tttcgcaaca agatttccca gaaagcgcac atcaacccaa
601 cactaatgtg cgataaccag ctcgatagaa atggaaattt catatggggt gagagaggat
661 accatgcaaa acgattcttc agcaactact ttgaaataat cgatccaaag caaggctaca
721 cccaatacga gacaagagtg gtaccaaatg ggtcacggaa acttgcaatc ggcaaactaa
781 tagtcccaac gaacttcgaa gttttaagag accagatgaa aggcgaaccg gtagaaccat
841 acccagtaac agtcgagtgt gtgagcaagt tacagggtga cttcgtccat gcatgttgtt
901 gtgtaacaac agaatcaggc gacccagtct tgtctgaaat aaaaatgcca accaaacacc
961 acctagtgat tggcaacagc ggcgatccaa agtacataga tctccctgag atcgaggaga
1021 ataaaatgta catagcaaaa gaaggttatt gttacatcaa catcttccta gctatgctag
1081 tgaatgtcaa ggaatcgcag gcaaaggagt tcacgaaagt tgttagggac aaactagttg
1141 gcgaacttgg caagtggccc actctgttag atgtagcaac cgcttgttat ttcctgaagg
1201 tattttaccc agacgttgct aacgccgaat tgccacgcat gttagtggat cataagacaa
1261 agataattca tgtcgttgat tcatatgggt cactgtcaac tggatatcac gtccttaaga
1321 caaacactgt ggaacaactc attaaattca cgagatgcaa tttggaatca agcttgaagc
1381 actaccgcgt cggaggaaca gagtgggagg acactcatgg agccagcaac atagatgatc
1441 cacagttgg
Claims (3)
1, a kind of method by Hc-Pro genetic transformation acquisition resisting turnip mosaic virus hot pickled mustard tube is characterized in that may further comprise the steps successively:
(1) callus that will induce through the cotyledon of 2 successive transfer culture is as transformation receptor;
(2) the Agrobacterium tumefaciems culture fluid and the callus that will have the Hc-Pro gene cultivated altogether;
(3) callus after will transforming constantly screens being added with on the antibiotic medium, breaks up then and plant regeneration.
2, Hc-Pro genetic transformation according to claim 1 obtains the method for resisting turnip mosaic virus hot pickled mustard tube, and it is characterized in that: 2 successive transfer culture in the step (1) may further comprise the steps successively:
(1) preserved szechuan pickle seeds is stirred sterilization 1min with running water flushing back with 75% medicinal alcohol, in the 1%NaClO aqueous solution, stir 15min behind the distilled water flushing 3 times, aseptic water washing 4-5 time, on the superclean bench seed is being sowed on the MS+0.1mg/L BA sowing medium, is cultivating in 23-25 ℃ of down dark place;
(2) the 16h/d continuous illumination under same temperature of germination back is cultivated;
(3) the aseptic seedling cotyledon of 8 days seedling ages being cut into 2mm left and right sides small pieces puts into callus inducing medium 23-25 ℃ of dark place and cultivates.
3, Hc-Pro genetic transformation according to claim 1 obtains the method for resisting turnip mosaic virus hot pickled mustard tube, and it is characterized in that: step (2) is carried out common cultivation 2 days under 24 ℃.
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| CN 200510062200 CN1839680A (en) | 2005-12-26 | 2005-12-26 | Method for transferring Sichuan pickle using TuMV Hc-Pro resistant gene |
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| CN 200510062200 CN1839680A (en) | 2005-12-26 | 2005-12-26 | Method for transferring Sichuan pickle using TuMV Hc-Pro resistant gene |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277376A (en) * | 2010-06-10 | 2011-12-14 | 叶锡东 | Gene transfer vector providing crop broad-spectrum virus resistance and containing pawpaw ringspot virus helper component protease gene and application thereof |
| CN102796739A (en) * | 2012-08-16 | 2012-11-28 | 北京农业生物技术研究中心 | Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant |
| CN103451197A (en) * | 2013-09-04 | 2013-12-18 | 北京农业生物技术研究中心 | TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA |
| CN106577219A (en) * | 2016-11-11 | 2017-04-26 | 浙江师范大学 | Plant transgenic seed screening method |
-
2005
- 2005-12-26 CN CN 200510062200 patent/CN1839680A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277376A (en) * | 2010-06-10 | 2011-12-14 | 叶锡东 | Gene transfer vector providing crop broad-spectrum virus resistance and containing pawpaw ringspot virus helper component protease gene and application thereof |
| CN102277376B (en) * | 2010-06-10 | 2013-09-25 | 叶锡东 | Gene transfer vector providing crop broad-spectrum virus resistance and containing pawpaw ringspot virus helper component protease gene and application thereof |
| CN102796739A (en) * | 2012-08-16 | 2012-11-28 | 北京农业生物技术研究中心 | Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant |
| CN102796739B (en) * | 2012-08-16 | 2014-02-19 | 北京农业生物技术研究中心 | Application of TuMV-CP gene fragment-mediated RNAi carrier in cultivation of anti-TuMV transgenic plant |
| CN103451197A (en) * | 2013-09-04 | 2013-12-18 | 北京农业生物技术研究中心 | TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA |
| CN103451197B (en) * | 2013-09-04 | 2015-09-02 | 北京农业生物技术研究中心 | The RNA of a kind of high resistance TuMV and the RNAi carrier of this RNA of coding |
| CN106577219A (en) * | 2016-11-11 | 2017-04-26 | 浙江师范大学 | Plant transgenic seed screening method |
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